A general object of some current therapeutic approaches is to interfere with, or otherwise modulate, gene expression.
One method for inhibiting the expression of specific genes involves the use of oligonucleotides, particularly oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence, known as antisense oligonucleotides. Several oligonucleotides are currently undergoing clinical trials for such use.
Oligonucleotides and their analogs can be designed to have particular properties. A number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness as therapeutic agents. Such modifications include those designed to increase binding affinity to a target strand, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide, to provide a mode of disruption (terminating event) once the oligonucleotide is bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide. Despite such modifications, the cellular uptake of oligomeric compounds remains poor.
Oligonucleotides have been formulated with various with transfection agents, including anionic and cationic lipids and polyamines, in an attempt to improve their ability to permeate biological membranes. Of the transfection agents used, polyethylenimines (PEI) are generally the most efficient and least expensive delivery vehicles. Kren, B. T.; Parashar, B.; Bandyopadhyay, P.; Chowdhury, N. R.; Chowdhury, J. R.; Steer, C. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10349-10354. It was observed, however, that, although complexes of excess PEI and oligonucleotide phosphorothioates were efficiently taken up by cells, the oligonucleotides failed to dissociate in the cytoplasm, resulting in no appreciable enhancement in the antisense activity of the oligonucleotides. Dheur, S.; Dias, N.; Van Aerschot, A.; Herdewijn, P.; Bettinger, T.; Remy, J.-S.; Helene, C.; Saison-Behmoaras, E. T. Antisense Nucleic Acid Drug Dev. 1999, 9, 515-525.
Another approach has been attempts to enhance the cellular uptake of oligomeric compounds comprising conjugating the compounds to amphipathic moieties such as amphipathic peptides. (PCT Publication WO 2004/016274).
Conjugation of terpyridine to PNA nucleotides have previously been shown to mediate cellular uptake of a PNA oligonucleotide as monitored by fluorescence. This process was suggested to be dependent on Zn2+ chelating. These experiments were carried out in HeLa cells using elevated concentrations (1-2.5 μM) of fluorophor labeled oligonucleotides added directly to the cell culture media. Addition of high concentrations of oligonucleotides usually leads to some uptake, probably through so called phase fluid endocytosis, as can be observed as a punctuated intracellular distribution, mostly located to endosomes. Interestingly conjugation of terpyridine to PNA led to increased nuclear localization of the oligo, indicating that oligonucleotide may be free to base pair with a potential target sequence, however, no such data was presented (Andreas Fuss, Andreas Schleifenbaum, Mareike Göritz, Andrew Riddel, Carsten Schultz, Roland Krämer, J. Am. Chem. Soc. 2006, 128(8), 5986-5987.)
A need therefore exists in the art for the development of means to improve the cellular uptake and cellular distribution of oligomeric compounds.
RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signalling (Mattick 2001; EMBO reports 2, 11: 986-991). Recent studies indicate that antisense transcription is a very common phenomenon in the mouse and human genomes (Okazaki et al. 2002; Nature 420: 563-573; Yelin et al. 2003, Nature Biotechnol.). Thus, antisense modulation of gene expression in eukaryotic cells, e.g. human cells appear to be a common regulatory mechanism. In light of this, the present invention provides a method for detection and functional analysis of non-coding antisense RNAs, as well as a method for detecting the overlapping regions between sense-antisense transcriptional units.